Project summary Respiratory viruses have a major impact on human health. Pneumonia is the primary cause of hospitalizations of the very young and a leading cause of death in older adults, and is most often caused by respiratory viruses. There is considerable variation in the susceptibility of humans to severe infection caused by these commonly encountered viruses, poorly understood at mechanistic levels. Further, the etiology of pneumonia remains enigmatic, with many infections caused by unidentified microbes other than the current suspects. Repeated exposures to respiratory viruses have a lasting impact on populations of memory lymphocytes that reside within the lung, contributing to the control of this local dynamic virome. This includes memory B cells. The shaping of B-cell memory through clonal selection and affinity maturation together leaves a signature of the individual's natural history of virome interactions. This imprint can be detected by sequencing the immunoglobulin variable-region gene (IgVRG) repertoire of lung B cells and analyzing their clonal structure statistically. We propose that identifying viruses recognized by lung B cells with the greatest affinity maturation will provide an unbiased window into the virome exerting immunological pressure on the lung. We will study lung B cells using a suite of specialized approaches including IgVRG repertoire analyses, an innovative Nojima culture system for growing B cell clones out of lung samples, and the newly developed multiplex VirScan technology for detecting viral specificity, in order to identify the virome that has exerted the most severe, recurrent, or chronic local immunological pressure on the human lung. These human lung virome analyses will be complemented by studies of how lung B cells and respiratory viruses interact causally, to test whether there are differences in memory B cells across compartments or conditions that determine the efficacy of local immune control of viruses in the lung. In the R61 phase, we will 1) use a mouse model to test the hypothesis that respiratory virome dynamics remodel lung B cells; 2) establish the Nojima B cell culture system for human lung B cells; 3) determine if human lungs contain virome-reactive B cells that can be detected by VirScan technology. If these proof-of-concept experiments are successful, then in the R33 phase we will test the hypotheses: 4) that the population-dynamic system comprising the human lung virome and lung-resident B cells is distinctive from other anatomical sites; 5) that there is heterogeneity within the human lung, such that central, conducting airway-rich regions of the lung recognize different viral antigens than do those from more peripheral regions of the same lung; 6) that long-term interactions between the human lung virome and resident B cells contribute to pneumonia susceptibility; 7) that lung memory B cells have a unique and specialized phenotype that provides rapid local antibody-mediated control of respiratory viruses. This study will leverage the lung resident B-cell compartment to provide novel insights into the lung virome and its relationships to pulmonary immunology and human health and disease.